The present invention relates to vaccines against Feline Infectious Peritonitis (FIP) prepared from the SPIKE (S) glycoprotein from the FIP virus whose major enhancing epitopes have been modified by mutagenesis.
These vaccines allow protection of cats vaccinated against FIP without causing in them the enhancement phenomenon which leads to an accelerated progression of the disease.
The Feline Infectious Peritonitis Virus (FIPV) is an enveloped, positive single-stranded RNA virus which, within the Coronaviridae family, belongs to the antigenic group which comprises the enteric feline coronavirus (FECV), the canine coronavirus (CCV), the pork transmissible gastronenteritis virus (TGEV) and the porcine respiratory coronavirus (PRCV) (Sanchez C. et al. Virology, 1990, 174, 410-417). This virus causes a disease which is complex and always fatal is cats, known as Feline Infectious Peritonitis (FIP). The FIP virus is defined among coronaviruses because it induces in cats the appearance of antibodies which enhance the infection by the virus and accelerate the progression of the disease. Cats having anti-FIPV neutralizing antibodies following a previous natural infection with this virus, following a passive transfer of antibody or following a vaccination, very frequently develop a disease which is much more intense and much more rapid than that in cats simply infected for the first time in the absence of specific antibodies (Pedersen N. and Boyle J., Am. J. Vet. Res. 1980, 41, 1868-876; Weiss R. et al., Comp. Immunol. Microb. Infect. Dis. 1981, 4, 175-189; Weiss R. et al., Am. J. Vet. Res. 1980, 41, 663-671). It is thought that the binding of the antibody-virus immune complexes to the Fc receptors present at the surface of macrophages constitutes the mechanism which enhances the acceleration of the entry of the virus into the cells and its rapid diffusion in the body (Porterfield, J. Advances in Virus research, 1986, 31, 335-355; Weiss et al. 1981). This enhancement phenomenon has been observed among the coronaviruses only with the FIP virus.
The FIP virus comprises three structural proteins. The largest in size is the xe2x80x9cSPIKExe2x80x9d or spicule (S) protein. This S protein is highly glycosylated and it is the one which induces in cats both neutralizing antibodies and enhancing antibodies. Studies carried out in vitro with neutralizing monoclonal antibodies directed against the FIP virus have shown that the major neutralizing epitopes are all situated on the S glycoprotein and that they correspond, to a large degree, to the epitopes involved in the enhancement phenomenon (Corapi W. et al., J. Virol. 1992, 66, 6695-6705; Olsen C. et al., J. Virol. 1992, 66, 956-965).
An effective vaccination against FIP should lead to the appearance of neutralizing antibodies without there being induction of enhancing antibodies. It has never been possible to develop such a vaccine up until now. The recombinant vaccines which do not contain the S glycoprotein can probably provide the best alternative for future FIP vaccines, but these antigens contribute only partially to the induction of the neutralizing response against the FIP virus. Of the three structural viral antigens, only the S glycoprotein is capable of inducing a substantial neutralizing response. Unfortunately, this glycoprotein also induces the concomitant appearance of enhancing antibodies. In spite of its importance in the induction of a good neutralizing response (and therefore in the protective response), the natural S glycoprotein appears to play an essential role in the FIP enhancement phenomenon and cannot therefore be used at the moment for the manufacture of vaccines meeting the criteria set out above.
The localization and characterization of the epitopes present on S and in particular those responsible for the neutralization and the enhancement is therefore necessary in order to determine the modifications to be made to the S glycoprotein (or to the gene which encodes this protein) in order to make it an effective immunogen for the vaccination of cats against FIP.
The nucleotide sequence and the protein sequence of the S glycoprotein of the FIP virus have been determined (de Groot R. et al. EP-A-0,264,979). This patent application does not teach how to identify the neutralizing epitopes and/or the enhancing epitopes on S. Neither does this document teach how to use the S sequence to manufacture a vaccine which is effective and nonenhancing against FIP.
Patent Application PCT WO-A-93/23421 claims the use of a truncated S glycoprotein or of a nucleic acid sequence encoding only a portion of S. In particular, the highly conserved region situated at the carboxy-terminal end of S (last 124 amino acids) is claimed for the preparation of a xe2x80x9cuniversalxe2x80x9d vaccine against coronaviruses. This document is very general and does not teach how to produce an FIP vaccine which does not induce enhancing antibodies in cats. The same is true of Patent Application PCT WO-A-93/23 422 which describes mixed constructs of FECV-FIPV chimeric S glycoprotein including the FIPV S fragments 542-597, 594-1454 or 651-1454.
Patent Application PCT WO-A-92/08487 claims the use of various peptides selected on the S proteins, or encoded by the S genes, of various FIPV virus strains, or by the FECV S gene sequence, for the diagnosis, treatment or prevention of FIP in cats. In particular, the 598-615 peptide of the S protein sequence of the FIPV virus strain 79-1146 is claimed for use in the form of a fusion protein with galactokinase, a recombinant protein capable of then being used for the diagnosis of anti-FIP antibodies in infected cats or as recombinant vaccine to induce protection against FIP in cats. Although envisaging variations in the sequences of the claimed peptides, this document does not teach precisely what the changes in the proposed sequences must be, and neither teaches how to produce a nonenhancing FIP vaccine, nor which of the S glycoprotein regions are involved in this phenomenon.
Patent Application GB-A-2,282,601, published after the priority date of the present application, proposes to produce a vaccine based on an S protein which is modified in order to avoid enhancement, by modification or deletion of at least one of the antigenic sites called D (corresponds to amino acids 496-524), A1 (corresponds to amino acids 531-555) and A2 (corresponds to amino acids 584-604), so as to make these sites antigenically inactive.
Great efforts have been made to identify the major antigenic sites present on the S proteins of the TGEV virus (Transmissible Gastro-Enteritis Virus) (Correa I. et al., J. Gen. Virol. 1990, 71, 271-279; Delmas B. et al., J. Gen. Virol. 1990, 71, 1313-1323), BCV (Bovine CoronaVirus) (Yoo D. et al., Virology 1991, 183, 91-98), MHV (Mouse Hepatitis Virus) (Takase-Yoden S. et al., Virus Res. 1990, 18, 99-108; Stuhler A. et al., J. Gen. Virol. 1991, 72, 1655-1658) and FIPV (Corapi W. et al., . Virol. 1992, 66, 6695-6705; Olsen, C. et al., J. Virol. 1992, 66, 956-965; Olsen C. et al., J. Gen. Virol. 1993, 74, 745-749). In all cases, multiple neutralizing domains were identified, and the immunodominant domains were generally localized on the S1 portion of the protein.
Studies relating specifically to the FIP virus have shown the existence on the S protein of epitopes which induce both a neutralizing response and an enhancing response with respect to infection with FIPV (Corapi W. et al., J. Virol. 1992, 66, 6695-6705; Olsen C. et al., J. Virol. 1992, 66, 956-965; Olsen C. et al., J. Gen. Virol. 1993, 74, 745-749). These same authors have shown that the neutralizing and enhancing monoclonal antibodies of anti-S specificity could be divided into 6 main groups according to their capacity to recognize different FIP virus strains and different mutants resistant to neutralization by these monoclonals (xe2x80x9cmarxe2x80x9d (monoclonal antibody resistant) mutants). However, the epitopes corresponding to the major antigenic regions on FIPV S have not been characterized. All the non-neutralizing monoclonal antibodies described by these authors (Olsen C. et al., J. Virol. 1992, 66, 956-965) are also nonenhancing in an in vitro enhancement test, which reinforces the hypothesis for a close relationship between neutralization and enhancement in the case of the FIP virus. The enhancement of viral infection by the antibodies occurs when the monocytes or macrophages are infected more effectively by the immune complexes, by a specific receptor-dependent endocytosis, than by the virus alone. In spite of all the studies performed on the antibody-dependent enhancement phenomenon, many questions remain unanswered. In particular, it is not known which specific viral components are responsible for the enhancement for each virus. Studies carried out up until now in FIPV indicate that the enhancement depends essentially on epitopes present on S (Olsen C. et al., 1993; Vennema H. et al., J. Virol. 1990, 64, 1407-1409).
Hohdatsu T. et al. (Arch. Virol. 1991, 120, 207-217) have found that anti-FIPV M monoclonal antibodies could induce an enhancement of the infection in vitro. This has not been confirmed in vivo by the studies performed with recombinants vaccinia/FIPV M and vaccinia/FIPV N. The immunization of cats with these two recombinants did not make it possible to observe an enhancement induced by either of these two proteins (Vennema H. et al. 1990). If M and N play a role in the enhancement, it is certainly at a level which is much lower than that played by S. During studies performed with the various viral systems where enhancement can be observed, a constant occurrence was observed: individual epitopes are capable of inducing both neutralizing antibodies and enhancing antibodies. This has been demonstrated for FIPV (Corapi W. et al., J. Virol. 1992, 66, 6695-6705; Olsen C. et al., J. Virol. 1992, 66, 956-965; Hohdatsu T. et al., Arch. Virol. 1991, 120, 207-217), for the dengue virus (Morens D. and Halstead S. J. Gen. Virol. 1990, 71, 2909-2917), and for HIV (Robinson W. Jr., J. Virol. 1991, 65, 4169-4176).
Recent results of the tests performed with experimental FIP vaccines appear to provide the most solid argument to date for the existence of a direct relationship between the enhancement observed in vitro and the accelerated disease in vivo in cats. The inoculation of cats with recombinants of the vaccinia virus expressing the S protein of the strain FIPV 79-1146 sensitizes the cats and induces after challenge an accelerated disease in the vaccinated cats compared with the nonvaccinated control cats (Vennema H. et al., J. Virol. 1990, 64, 1407-1409). The inoculation of vaccinia recombinants expressing either the M protein, or the N protein, need not predispose the cats to an accelerated disease. These in vivo results are to be taken in parallel with the in vitro results demonstrating a predominant localization of the enhancing epitopes on S (Corapi W. et al., J. Virol. 1992, 66, 6695-6705; Olsen C. et al., J. Virol. 1992, 66, 956-965). Furthermore, recent experiments performed in order to study the efficacy of another candidate vaccine for FIP have demonstrated a statistically significant association between the capacity of a cat serum to induce an enhancement in vitro and the development in the same cat of an accelerated disease (Olsen C., Vet. Microb. 1993, 36, 1-37).
The subject of the present invention is the characterization of the epitopes involved in the enhancement of the FIP virus infection. The precise knowledge of the molecular structures responsible for the enhancement mechanism makes it possible to design antigens which do not induce the appearance of enhancing antibodies. These antigens are the essential components of an effective FIP vaccine.
Surprisingly, it has been discovered, by analyzing the sequence of the S gene of mutant FIPV viruses resistant to neutralization with neutralizing and enhancing monoclonal antibodies, or resistant to monoclonal antibodies which are only neutralizing and not enhancing, that it was possible to sidestep the mechanism of induction of enhancement by the S glycoprotein. Two major antigenic sites have been characterized with the monoclonal antibodies studied: A1 and A2. These sites are both surprisingly situated in the same region of the S protein. It appears that the strongly neutralizing and enhancing antibodies recognize both sites at the same time. This information suggests that the simultaneous binding of the two epitopes by the same antibody plays a direct role in the enhancement. Indeed, in parallel with this first discovery, it has been discovered that the neutralizing, but not enhancing, antibodies recognize only the A2 site. Enhancement could therefore be due to a confirmational modification by the coming together of the two epitopes A1 and A2. The A2 region includes amino acids 637-662 on the protein sequence of S (De Groot R. et al., J. Gen. Virol. 1987, 68, 2639-2646). The hydrophilic nature of this region and the fact that the 3 monoclonal antibodies tested all recognize this small domain suggest that A2 is a dominant neutralizing epitope of the S protein. Moreover, the close homology observed between the A1 site and a portion of the Aa subsite identified on the TGEV S protein (Gebauer F. et al., Virology 1991, 183, 225-238) suggests that A1, which comprises amino acids 562-598 must also be an important neutralizing epitope for the FIPV virus.
The coming together or the simultaneous binding, by the same antibody, of the A1 and A2 sites is necessary in order to induce enhancement through antibodies.
The subject of the present invention is the modification, by genetic engineering, of the sequence of the FIPV S gene in the region of the A1 and/or A2 sites, in particular in order to modify at least one of both sites, preferably to modify the A1 site so that the protein expressed presents an epitope modified so that the protein no longer induces enhancing antibodies and/or the A2 site. The A1 region can be modified in various ways, by means well known to persons skilled in the art. The A1 or A2 sites can be modified independently or simultaneously.
The modification of the A2 site may consist in a modification, such as a complete deletion, leading to a loss of antigenicity of the site, but one preferes that, as for the A1 site, the modification expresses an epitope which is modified so that the protein no more induces enhancing antibodies.
The A1 region has a common part with the so-called xe2x80x9cA2xe2x80x9d region in patent application GB-A-2,282,601 (WO-A-95/07987) cited above, and contrary to the invention it provides for mutations (modifications) or deletions which cause a loss of antigenicity of the modified or deleted region.
The subject of the invention is in particular a nucleotide sequence comprising the complete FIPV S gene, having at least one modification, preferably a mutation and/or limited deletion, in the antigenic A2 region which encoder amino acids 637 to 662 and/or in the antigenic A1 region which encodes amino acids 562 to 598, with the exception of total deletion or an important mutation or deletion having the same consequences than a total deletion, say a loss of antigenicity of the modified region.
The present application is now limited to that part of the invention which concerns the modifications which allow the suppression of the enhancing antibodies induction without modification of the antigenicity, at least for the A1 site.
Of course, the expression nucleotide sequence comprising the complete FIPV S gene covers the types 1 and 2 FIPV strains as well as the variants and the sequences which exhibit secondary variations, that is to say which do not affect the immunogenicity of the S protein, which also covers secondary mutations and deletions outside the A1 and A2 sides. Preferably, the variations in the sequence must not modify the functionality of the S glycoprotein.
This therefore includes the sequences having a high degree of homology with the previous sequences, including when the degeneracy of the genetic code is taken into account, this homology being sufficiently high so that the expressed polypeptide makes it possible to induce an effective vaccinal protection.
Limited deletion is understood to mean preferably a point deletion (corresponding to 1 amino acid) or a microdeletion (up to 6 amino acids).
Mutations or deletions of the codons encoding the cysteines situated in A1 and A2 will be avoided in general.
In addition, mutations and secondly point deletions (except Cys) will be preferred to more extensive mutations and deletions.
For the A1 site, the modifications comprise a minima a mutation for at least one and, preferably, for both of the codons encoding Asp 568 and Asp 591 in order to have any other amino acid at these positions. Provided that amino acids 568 and 591 are not Asp, any other amino acid in the 562-598 region can be substituted for the natural amino acid in the position considered.
The modifications of the A1 site also comprise limited deletions of this region comprising amino acids 568 and/or 591.
For the A2 site, the modifications comprises a minima a mutation for at least one and, preferably, for the three codons encoding Asp 643, Arg 649 and Arg 656 in order to have any other amino acid at these positions. The modifications of the A2 site also comprise the total deletion/partial deletions of this region comprising amino acids 643, 649 and/or 656.
The subject of the present invention is also the use of the FIPV genes thus modified for the in vitro expression of recombinant FIPV S proteins and for the preparation of purified subunit vaccines for the vaccination of cats against FIP.
The subject of the present invention is also the use of the FIPV S genes thus modified for the construction of recombinant viral vectors expressing these modified genes. These viral vectors may be replicative or nonreplicative recombinant viruses and more particularly poxviruses (example: vaccinia virus and its derivatives, canarypox virus and the like), herpesviruses (in particular feline herpesvirus), or adenoviruses.
The subject of the present invention is the preparation of vaccines against FIP with these recombinant viruses.
The subject of the present invention is also the immunization of cats against FIP with plasmids containing the FIPV S genes, modified according to the present invention, and placed under the control of a strong promoter (for example HCMV IE, SV40, and the like) and of regulatory signals for transcription and translation. The plasmids are present in a vehicle capable of allowing direct injection into cats, especially via the intramuscular route. They are especially naked plasmids as described in International Patent Application WO 90/11092.
The subject of the present invention is finally the preparation of vaccines against FIP comprising one (or more) FIPV S protein(s), modified according to the present invention, preferably combined with other FIPV virus proteins such as for example the M protein.
Another vaccinal solution consists in using cells (in particular of feline origin) constitutively expressing the S glycoprotein according to the invention.